The overall goal of this proposal is to unravel functional and molecular defects of neuromuscular synaptic transmission at the level of the neuromuscular junction in Amyotrophic Lateral Sclerosis (ALS). We propose to use ALS-related Fused-in sarcoma (FUS) as a prototype protein to identify neuromuscular junction (NMJ) targets of disease and to unravel the mechanisms by which ALS-linked FUS mutations alter the function of the NMJ, which is a prominent feature of ALS. Specifically, we propose to identify critical synaptic proteins targeted by mutant FUS in the terminal axon of the pre-synaptic motor neuron at NMJ. With the use of human iPS cells-derived motor neuron/muscle co- cultures from different sub-groups of patients (mutant FUS, SOD1, C9Orf72 and sporadic), we will in addition compare synaptic targets impaired by mutant FUS with those potentially impaired by other ALS-related genetic and environmental stressors. Ultimately, we will identify FUS specific vs. common ALS NMJ targets. Our plan is to employ a systematic, step-based and collaborative research approach that uses multiple research tools and disease models, each with its own characteristics and strengths. We will combine the expertise of the Pasinelli laboratory in studying molecular pathogenic mechanisms in ALS in disease relevant cells/systems with the experience of the Maragakis' lab in using and characterizing iPS cells to: (1) Investigate the cellular and molecular mechanisms of mutant FUS-induced dysfunction of the NMJ; (2) Identify FUS- specific and common targets of NMJ dysfunction in ALS patients, ultimately for biomarker identification and drug discovery.